Try to add DTT in sample buffer in parallel to DTT+0.5M Urea, this might help to unmask the antigenic epitope.

Second, just 3+3 flag tag (N+C terminal) might not discriminate your flag tagged-protein X from endogenous protein X unless you use 15% gel and run SDS-PAGE longer time. You might try immunoprecipitate Flag-Protein-X and then do Western using antibody for protein-X (this will help to identify whether your endogenous protein and flag tagged protein are in the same band. OR there may be no endogenous protein and what you see might be flag tagged (if you have an untransfected control, you'll be able to find this out).

[Second suggestion is based on your statement: "Using an anti-FLAG (recognizes 3X FLAG peptide) I was able to see the correct size of my tagged protein (via Western Blot) ~ 53 kDa (a nice bright band). When I used the antibody against Protein X I was NOT able to see the "doublet" band only the endogenous protein band (~50 kDa)."].

Your antibody might recognize the extreme N- or C-terminus of the protein (in fact, many antibodies do), which is now masked by the tag.

Good point! Thank you Ralf.

But I know the Antibody we have was raised against a peptide sequence that is internal to the protein (polyclonal Ab). I have tried different bleeds with the same result.

You could try denaturing rigorously, for instance boil at 90 degrees in the loading buffer containing b-mercap for 5-10 minutes, spin briefly and then load. I would run a lane with the untagged version in parallel just to make sure, in a particular experiment, everything is going technically correct. If nothing works, look for a different primary :(

Thanks! Yes I forgot to add I also tried various Antibodies for Protein X all to different sites on the protein and still nothing. I just can't believe there is a coincidence that the FLAG Ab is recognizing something at the expected size. I hope it's not.

I will try the heavy denaturing. Thanks.

By the way, seems other people are having the same problem:

https://www.researchgate.net/post/Has_anyone_experienced_an_inability_for_protein_specific_antibodies_to_bind_their_target_when_the_protein_has_a_Flag_Tag

Are your gel conditions such that you could see the difference in size if it were there?

Try to add DTT in sample buffer in parallel to DTT+0.5M Urea, this might help to unmask the antigenic epitope.

Second, just 3+3 flag tag (N+C terminal) might not discriminate your flag tagged-protein X from endogenous protein X unless you use 15% gel and run SDS-PAGE longer time. You might try immunoprecipitate Flag-Protein-X and then do Western using antibody for protein-X (this will help to identify whether your endogenous protein and flag tagged protein are in the same band. OR there may be no endogenous protein and what you see might be flag tagged (if you have an untransfected control, you'll be able to find this out).

[Second suggestion is based on your statement: "Using an anti-FLAG (recognizes 3X FLAG peptide) I was able to see the correct size of my tagged protein (via Western Blot) ~ 53 kDa (a nice bright band). When I used the antibody against Protein X I was NOT able to see the "doublet" band only the endogenous protein band (~50 kDa)."].

It could be possibile that the protein is cleaved and so you loose the tag?

Regards..

Francesco

You could try boiling your entire blot in PBS in a microwave oven for about 5 minutes. We used this technique to unmask an epitope of a peptide which was not detectable before, even though we added beta-ME and boiled the samples, and it worked.

Normally, when I overexpress tagged proteins in eukaryotic cells, the proteins can be detected by immunoblotting both with anti-tag antibodies and anti-protein antibodies. But in your case, the endogenous protein can be detected but not the overexpressed protein (tagged one) with the same antibody, the possible explanation could be that the expression of FLAG tags might mask the antigen (1st scenario) or the antigen is not there at all (the second scenario). But since you are using denaturing conditions, the 1st scenario would unlikely seem to happen. I will suggest you to proceed with a simple experiment, just overexpress your protein without any tag (if you have the same sequence in a different backbone vector) and analyze the sample via immunoblotting (the antigen of the antibody might be missing in your protein). But if you have to start from the beginning (to get the sequence in a different backbone vector), you might also proceed with Mass spectrometery and analyse the purified protein sequence. I hope these suggestions will help you to narrow down your concentration on the real problem.

Hi, I am also having the same problem. My overexpressing protein is tagged by 6x His on the C terminus. I can detect the overxpressing protein using 6x His Ab but not the protein specific Ab. My protein specific Ab is probing to the extreme C-terminus. Therefore, the epitope which the protein specific Ab recognize may be masked by the tag as you guys suggested above.

But actually how the tag can still masking the epitope nearby if we are running a denature SDS-PAGE. Is it because the degeneration step is not go enough? Anyone know? Thanks a lot!

To Linda Schauenburg:

Thanks for your suggestion. Are you blots from denature SDS-PAGE?

Even though you run the gel under denaturing conditions, there will be some refolding during the transfer procedure, so there is some structure (not necessarily correct structure) on the blot.  Have you expressed your tagged protein in a negative cell (bacteria) and probed for FLAG and native protein.  Since you have what you say is a good antibody against your protein, what are you trying to accomplish with the epitope?  You may have to bite the bullet and try a different epitope tag.

Hi, I have the exactly same problem and wonder if you have figured out a reason or managed to detect your protein?

Lan,

We were not able to resolve this matter. BUT we still submitted for mass spec analysis (the pulldown) and did identify our protein of interest. If we go back to resolving this problem I will update this discussion. All the best.